DDT inhibits the functional activation of murine macrophages and decreases resistance to infection by Mycobacterium microti.

DDT is still widely used in several parts of the world to control malaria, typhoid and dengue vectors, even though its use was banned in many countries based on toxicity data in wild life species. DDT has been shown to have immunotoxic effects in mice and to increase susceptibility to intracellular pathogens such as Mycobacterium leprae. However, little is known about the mechanisms underlying this effect. Activated macrophages play an important defensive role against intracellular pathogens, therefore our objective was to evaluate the effect of in vitro exposure to technical grade DDT (a mixture of three forms: 1,1,1-thricloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) (85%), o,p'-DDT (15%) and o,o'-DDT (trace amounts)), p,p'-DDT, 1,1-dicloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane on the functional activation of J774A.1 macrophages and their capability to limit growth of intracellular pathogens, using Mycobacterium microti as a model. We evaluated cytotoxicity and the effect on cell proliferation of 2.5, 5.0 and 10 microg/ml of DDT compounds. Functional macrophage activity (NO(*) and O(2)(-) production, and mRNA expression of TNF-alpha, IL-1beta and iNO synthase) and the ability of treated cells to limit infection by M. microti in IFN-gamma-activated macrophages were evaluated in cells exposed to 2.5 microg/ml of DDT compounds. Doses of 5 and 10 microg/ml induced direct cytotoxic effects precluding meaningful analysis of the above parameters, whereas 2.5 microg/ml of all DDT compounds inhibited macrophage activity and reduced their ability to limit the intracellular growth of M. microti without inducing cytotoxicity. Technical grade DDT and p,p'-DDE were the more potent compounds. Therefore, exposure to DDT compounds could represent an important risk for infection development by those intracellular pathogens against which NO(*) and/or O(2)(-) production represent the main immune protective mechanism.

The flagellar fraction of Trypanosoma cruzi depleted of an immunosuppressive antigen enhances protection to infection and elicits spontaneous T cell responses.

The flagellar fraction (FF) of Trypanosoma cruzi can be separated by immunoaffinity chromatography in two fractions with balanced but opposite immunological effects. The immunoaffinity purified fraction has immunosuppressive activity mediated at least partially by TGF-beta (Hansen et al., submitted). Here we report that the fraction depleted of immunosuppresive antigens (FT) administered with iscom-matrix as adjuvant provides enhanced protection to an infection challenge in immunized mice. In vitro, the FT but not the FF stimulated resident peritoneal cells to produce IL-1 and IL-6. In immunized mice, the FT elicited higher levels of antigen-specific IgG2a than the FF as well as broader recognition of T. cruzi antigens. Splenocytes from mice immunized with FT proliferated spontaneously in vitro and secreted TH1 and TH2 cytokines. The protection provided by FT correlates with its capacity to enhance the secretion of IFN-gamma. We postulate that immunosuppressive antigens present in the FF prevent the development of memory cells secreting IFN-gamma through a TGF-beta dependent mechanism.

Cytokines in mycobacterial infections: in vitro and ex vivo studies.

Different species of mycobacteria differ in their capacity to induce the production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes in vitro. Whereas M. tuberculosis is a potent inducer of TNF-alpha, M. leprae is much less potent. TNF-alpha production is found to be associated with the availability of H2O2 generated by activated monocytes, as superoxide enhancing H2O2 concentration increases and catalase degrading H2O2 decreases TNF-alpha production. Furthermore, M. kansasii with high intrinsic catalase induce less TNF-alpha than mycobacteria with low intrinsic catalase. In vitro infection of monocytes with M. tuberculosis leads to an impairment of the antigen-presenting capacity, as determined by a reduction of antigen-induced T cell proliferation and interferon gamma (IFN-gamma) production. Of crucial importance is this impairment is the M. tuberculosis-induced down-modulation of MHC class II antigens. The role of TNF-alpha in vivo is reflected in patients with various forms of leprosy. In skin lesions of lepromatous leprosy patients TNF-alpha, interleukin 1 beta (IL-1 beta), and INF-gamma production are found to be rare, whereas these cytokines are well expressed in skin lesions of patients with tuberculoid leprosy. After multidrug chemotherapy an increase of local cytokine production is found. Taken together, these findings suggest that components of mycobacteria may interfere with local cell-mediated immune reactions in vivo. The molecular mechanisms involved in these local responses need to be defined.

Differential production of interleukin 1 (IL-1), IL-6, tumor necrosis factor, and IL-1 receptor antagonist by human monocytes stimulated with Mycobacterium leprae and M. bovis BCG.

Human blood monocytes cultured in various serum conditions were stimulated with Mycobacterium leprae or M. bovis BCG and their cytokine-inducing abilities were compared. BCG, either live or killed, induced production of interleukin 1 (IL-1), IL-6, tumor necrosis factor (TNF), and IL-1 receptor antagonist (IL-1ra). Live BCG at a lower bacterial number was more potent than killed BCG in the induction of IL-6 and TNF. In contrast to BCG, killed M. leprae induced few cytokines except for IL-1ra. Similar results were obtained when monocytes were cultured in the presence of untreated or heat-inactivated fetal bovine serum (FBS). When FBS and human serum (HS) were compared and the effect of heat inactivation was investigated, monocytes in HS produced the most cytokines, then those in FBS, irrespective of heat inactivation, and those in heat-inactivated HS produced the least cytokines. There were no differences between live and killed M. leprae, and BCG were far more potent than M. leprae in all of our experimental conditions, indicating that the poor cytokine (IL-1, IL-6 and TNF)-inducing ability of M. leprae was not due to their viability. Cytokine production was partially in parallel with the phagocytosis of the mycobacteria. These results suggest that M. leprae favor their infection by evoking little host reaction through the induction of only low levels of immunostimulatory or proinflammatory cytokines but a substantial amount of immunosuppressive cytokine.

[Monokine production by mouse peritoneal macrophages after phagocytosis of mycobacteria].

It is well known that monokines, IL-1 (Interleukin-1) and TNF (Tumor Necrosis Factor), are produced by macrophages after stimulated with various agents. These cytokines are involved in various aspects of the inflammatory process and immunological response in addition to their original activities to proliferate T lymphocytes and causing tumor necrosis, respectively. Recently, there have been reported that IL-1 and TNF also play an important role in mycobacterial infections such as granuloma formation. In the present study, IL-1 and TNF productions were observed by mouse peritoneal exudate and resident macrophages after incubation with heat-killed M. lepraemurium and M. avium in vitro. The production was enhanced by phagocytosis of these mycobacteria in a dose dependent manner, and the time course of the production was maximum within 24 hr after phagocytosis of these mycobacteria. It was also shown of morphological changes and enhanced glucose consumption in media by these macrophages. Above results suggest that phagocytosis of mycobacteria by macrophages leads to monokine production, which would not only causes well known immunological reactions but also makes characteristic phenomena to be observed in mycobacterial infections.

Rat Schwann cells produce interleukin-1.

There is increasing evidence that Schwann cells of peripheral nerves may be able to function as accessory cells, interacting with the immune system in T cell-mediated immune responses, by expression of the major histocompatibility complex (MHC) class II molecules. In addition to MHC class II-associated presentation of antigen to T lymphocytes, the release of a co-stimulatory factor, interleukin-1 (IL-1), is an essential function of accessory cells for T cell activation. In this study, we investigated if Schwann cells were able to produce IL-1. Purified cultures of neonatal and adult rat Schwann cells were incubated with various stimulatory agents. Supernatants and cell lysates were collected from these cultures and IL-1 activity was assayed. Both neonatal and adult rat Schwann cells produced IL-1 activity in response to bacterial antigens and the IL-1 activity was often higher in the cell lysate than in the supernatant. When stimulated neonatal or adult rat Schwann cells were examined with antibody against IL-1, strong immunolabelling was seen intracellularly, but no IL-1 was detected on the cell surface. Since IL-1 plays an important role in the initiation of immune responses, these observations support the view that Schwann cells may function as antigen-presenting cells, thereby taking part in neuroimmunological responses within peripheral nerves.

Interleukin-1 beta production by monocytes from leprosy patients.

The cause responsible for the lack of an efficient cell-mediated immunity or a delayed type hypersensitivity to M. leprae in lepromatous patients is poorly understood. But the resistance to M. leprae infection in humans is likely mediated by the activated macrophages to present M. leprae antigen to T cells for cell-mediated immunity. Phenolic glycolipid-I (PGL-I) is a M. leprae-specific antigen and is supposed to play a significant role in the long lasting unresponsiveness in lepromatous leprosy. In this study, IL-1 activities were tested among leprosy patients to evaluate monocyte function and the role of IL-1 in the immunosuppression in leprosy. We found that peripheral blood mononuclear cells (PBMCs) from tuberculoid patients were strongly reactive to M. leprae (mean cpm; 28,853 +/- 28,916), but the proliferative responses of PBMCs from lepromatous patients (mean cpm; 6,051 +/- 803) were significantly lower. IL-1 concentration in culture supernatant of monocytes from lepromatous patients was similar to that from tuberculoid patients with stimulation of M. leprae (lepromatous: 1,014 +/- 637 pg/ml, tuberculoid: 1,012 +/- 167 pg/ml) or lipopolysaccharides (IPS) (lepromatous: 3,479 +/- 2,188 pg/ml, tuberculoid: 4,246 +/- 2,432 pg/ml). The IL-1 concentration is sera from lepromatous patients (42 +/- 30 pg/ml) tended to be higher than those from tuberculoid patients (28 +/- 69 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic defects in leprosy patients. II. Interleukin 1, interleukin 2, and interferon production in leprosy patients.

The capabilities of monocytes and lymphocytes in peripheral blood mononuclear leukocytes (PBML) to produce interleukin-1 (IL-1), IL-2, and interferon (IFN), respectively, were evaluated in various types and treatments of leprosy patients. IL-1 production in response to lipopolysaccharide was significantly lower in LL, BL, BB, and BT patients than in normal controls. However, there were no differences in IL-1 levels between TT patients and normal controls. The percentages of nonspecific-esterase-positive cells adhering to the plastic surfaces were not different in LL, BB and TT patients when compared to normal controls. However, they were significantly higher in BT and BL patients than in normal controls. When PBML from leprosy patients were stimulated with concanavalin-A (ConA) for IL-2 production, there were no differences in the IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients compared to normal controls. Similar results were obtained when PBML were stimulated with phytohemagglutinin-P (PHA-P). However, when purified protein derivative (PPD) was used as the stimulating agent, there were significantly lower IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients when compared to normal controls. There were also lower IL-2 levels in untreated BL/LL and BT/TT patients compared to treated BL/LL and BT/TT patients, respectively. PBML were stimulated with PHA-P or ConA for IFN production.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 1 and prostaglandin production by cells of the mononuclear phagocyte system isolated from mycobacterial granulomas.

A study has been made of the activity of interleukin 1 (IL-1) and prostaglandins (PGs) in the culture supernatants from unstimulated and lipopolysaccharide (LPS)-stimulated mycobacteria-induced granuloma cells. Both epithelioid cells from bacillus Calmette-Guerin (BCG)-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas, separated on a fluorescence-activated cell sorter using monoclonal antibody specific to guinea pig macrophages, spontaneously secreted low levels of IL-1 (assayed by thymocyte comitogenic and fibroblast mitogenic activities) into culture supernatants. However, culture supernatants from LPS-stimulated epithelioid cells showed significantly higher IL-1 activity than those from unstimulated cells. In contrast, LPS stimulation of M. leprae granuloma macrophages failed to enhance IL-1 production. Nevertheless, IL-1 activity in the culture supernatants from stimulated mycobacterial granuloma cells of both types was much lower than that from LPS-stimulated peritoneal exudate macrophage culture supernatants. There was no detectable amount of prostaglandin E2 (PGE2) in the culture supernatants from both unstimulated and LPS-stimulated BCG- and M. leprae-induced granuloma cells in comparison to much higher levels of PGE2 produced by unstimulated (0.28-6.2 ng/ml) or LPS-stimulated (greater than 15 ng/ml) peritoneal exudate macrophages. However, BCG granuloma cells either secreted prostaglandin F2 alpha (PGF2 alpha) spontaneously or produced comparable levels of PGF2 alpha to those from peritoneal exudate macrophages on stimulation, while M. leprae granuloma macrophages produced much lower levels of PGF2 alpha.

Mycobacterium leprae fails to stimulate phagocytic cell superoxide anion generation.

Mycobacterium leprae is an intracellular pathogen that is ingested by and proliferates within cells of the monocyte/macrophage series. Mechanisms by which intracellular pathogens resist destruction may involve failure to elicit a phagocyte "respiratory burst" or resistance to toxic oxygen derivatives and lysosomal enzymes. We have studied the ability of M. leprae and Mycobacterium bovis BCG to stimulate the generation of superoxide anion (O2-) in vitro by human blood neutrophils and monocytes and murine peritoneal macrophages. M. leprae bacteria failed to stimulate significant O2- release except at high bacteria-to-cell ratios (greater than 50:1) whether or not they were pretreated with normal serum or serum from patients with lepromatous leprosy. Either viable or irradiated BCG; on the other hand, stimulated the three cell types to release significant amounts of O2- when challenged with as few as 10 organisms per cell. Serum pretreatment enhanced the release of O2- by the three cell types. Preincubation for 18 h with viable M. leprae did not inhibit the ability of monocytes to respond with an oxidative burst to phagocytic stimuli. The failure of M. leprae to stimulate phagocyte O2- generation may be an important factor in its pathogenicity.

Defective cell-mediated immunity in leprosy: failure of T cells from lepromatous leprosy patients to respond to Mycobacterium leprae is associated with defective expression of interleukin 2 receptors and is not reconstituted by interleukin 2.

Patients with lepromatous leprosy (LL) but not borderline tuberculoid leprosy (BT) have defective cell-mediated immune responses to Mycobacterium leprae, despite normal responses to other stimuli, as judged by in vivo skin testing and in vitro lymphocyte transformation. To investigate the basis of the immune defect in LL patients, we studied the ability of patient mononuclear leukocytes to produce interleukin 1 (IL 1) and interleukin 2 (IL 2) upon stimulation with M. leprae, and determined the ability of exogenous IL 1 and IL 2 to reconstitute the LL patient response to this antigen in vitro. Equal numbers of adherent non-T cells from LL and BT patients produced similar amounts of IL 1 upon challenge with M. leprae, and addition of IL 1 to the culture medium failed to reconstitute the response of lymphocytes from LL patients to M. leprae. On the other hand, T cells of LL patients failed to express receptors for IL 2 or to produce IL 2 in response to M. leprae, whereas similarly treated T cells of BT patients both expressed IL 2 receptors and produced IL 2. Finally, recombinant human IL 2 purified to homogeneity as well as crude supernatants of mitogen-activated lymphocytes failed to reconstitute the response of LL patients to M. leprae. These results suggest that T cells of LL patients fail to respond to M. leprae despite an ability to produce IL 1 and that their failure to express receptors for IL 2 may explain both defective proliferation and the failure of exogenous IL 2 to reconstitute the response.

Dissociation between interleukin-1 and interleukin-2 production in proliferative response to microbial antigens: restorative effect of exogenous interleukin-2.

The relationship between the production of interleukin-1 (IL-1) and interleukin-2 (IL-2) after stimulation of human mononuclear cells within an antigenic extract from Candida albicans was analyzed in both responder and nonresponder donors. Culture supernatants from responders contained both IL-1 and IL-2 activity, whereas the supernatants from nonresponders contained only IL-1 and no appreciable IL-2. However, the addition of exogenous IL-2 to nonresponder cultures restored the normal proliferative response. Similar observations were made when cells from mice infected intravenously with high doses of Mycobacterium bovis BCG were cultured; these cells showed a marked impairment of the proliferative response to purified protein derivative. Spleen cells from BCG-induced unresponsive mice failed to produce IL-2 despite the fact that normal IL-1 activity was present in the culture. Again, the addition of exogenous IL-2 fully reversed the proliferative unresponsiveness. Thus, the presence of IL-1 does not necessarily induce production of IL-2, and the proliferative unresponsiveness is therefore due to a primary lack of IL-2.

Interleukin 1 production by peripheral blood mononuclear cells from leprosy patients.

Quantitation of interleukin 1 production by adherent mononuclear cells from peripheral blood was performed in patients with tuberculoid and lepromatous forms of leprosy. Cells from patients with tuberculoid leprosy either secreted interleukin 1 spontaneously or produced amounts within the normal range in response to lipopolysaccharide stimulation. Conversely, stimulated cells from lepromatous patients failed to produce interleukin 1 in 5 of 13 (38.5%) cases.

Reversal by interleukin-2 of the T cell unresponsiveness of lepromatous leprosy to Mycobacterium leprae.

In some subjects Mycobacterium leprae causes disseminated (lepromatous) disease. Such subjects show both in vivo and in vitro deficient T cell responses to M. leprae, but not to other antigens. We have recently shown that lepromatous peripheral blood mononuclear cells (PBMC) failed to produce interleukin 2 (IL-2) in response to M. leprae and that T cell-conditioned media (TCM) can reverse the T cell unresponsiveness in a majority of lepromatous leprosy patients (Haregewoin et al. 1983). Here we show that highly purified and recombinant IL-2 had effects similar to TCM. On the other hand, lepromatous PBMC produced IL-1, and IL-1 had no restorative effect. These findings provide further evidence that the unresponsiveness in lepromatous leprosy often results from a deficiency in IL-2 production. After initial stimulation with TCM + M. leprae, lepromatous PBMC could be restimulated with M. leprae alone, providing clear evidence that M. leprae-reactive lymphocytes were generated in the presence of TCM. The present findings are discussed in relation to the possible mechanisms involved in the failure of IL-2 production. If our findings can be reproduced in vivo, IL-2 may offer a novel approach to therapy in lepromatous leprosy.